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Potato X virus resistant attenuated vaccine as well as preparation method and application thereof

An attenuated vaccine and potato technology, which is applied in the field of plant anti-virus genetic engineering, can solve the problems of mutant virulent strains, few types of attenuated strains, and inconvenient application, so as to reduce damage, delay plant disease, and stabilize the effect. Effect

Active Publication Date: 2020-03-17
SHANDONG AGRICULTURAL UNIVERSITY +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The key factors that limit the wide application of cross-protection are firstly that there are not many types of attenuated strains available at present, and the application is inconvenient; is an amino acid mutation
These attenuated strains or mutants have the risk of mutating into virulent strains when used to prevent and treat viral diseases

Method used

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  • Potato X virus resistant attenuated vaccine as well as preparation method and application thereof
  • Potato X virus resistant attenuated vaccine as well as preparation method and application thereof
  • Potato X virus resistant attenuated vaccine as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Example 1 Construction of multi-site TVBMV attenuated mutants

[0044] 1. Construction of Infectious Clones of Tobacco Vein Mosaic Virus

[0045]Using the RNA of tobacco vein mosaic virus as a template, reverse transcription was performed with random primers. According to the restriction enzyme digestion map of the existing tobacco vein mosaic virus genome, it can be amplified in three parts, and assembled into a TVBMV full-length cDNA clone after enzyme digestion. First, the 35S promoter was fused to the upstream of the fragment from the 5′ untranslated region of TVBMV to the Nru I restriction site of the HC-pro gene by Overlap-PCR, and the inventor named the fragment p35S-HC; PCR amplification of HC- The fragment between the Nru I restriction site of the pro gene and the 6K2 Xho I restriction site was named pHC-6K2 by the inventor; the 6K2Xho I restriction site was amplified by PCR to the tail of the poly(A) A fragment, which the inventors named p6K2-polyA (as figu...

Embodiment 2

[0063] Example 2 Amplification of Potato X Virus Related Gene Fragments and Construction of Attenuated Vaccine

[0064] 1. Amplification of potato X virus-related gene fragments

[0065] Using the PVX-1985 genome as a template, RT-PCR was used to amplify each gene fragment. The embodiments provided by the present invention are all in accordance with conventional experimental conditions, and the primer sequences used are as follows:

[0066] Table 2 PVX gene fragment amplification primer sequences

[0067]

[0068]

[0069] Among them, primers 1 and 2 are used to amplify the Rd1 region of PVX, primers 3 and 4 are used to amplify the Rd2 region of PVX, primers 5 and 6 are used to amplify the Rd3 region of PVX, and primers 7 and 8 are used to amplify the PVX region. The CP region, primers 9 and 10 were used to amplify the TGB region of PVX. The nucleotide sequence of the amplified Rd1 gene is shown in Seq ID No.17, the nucleotide sequence of the Rd2 gene is shown in Seq ...

Embodiment 3

[0083] Example 3: Chimeric virus inoculated plants

[0084] The chimeric virus obtained in Example 2 was transformed into Agrobacterium GV3101. After verification by colony PCR, recombinant bacteria were obtained. Then single spot was inoculated in liquid LB medium containing kanamycin (50 μg / mL), rifamycin (50 μg / mL) and tetracycline (50 μg / mL). Add 500 μL of bacterial liquid to 5 mL of LB medium containing 10 mmol / L 2-(N-morpholine)-ethylsulfonic acid (MES), 20 μmol / L acetosyringone (AS) and the above three antibiotics, at 28°C Shake culture to logarithmic growth phase.

[0085] Cells were collected by centrifugation and resuspended in 10 mmol / L MgCl 2 , 10mmol / LMES, 150μmol / L AS, adjust the concentration to make the OD 600 is about 0.5, and let stand for 3 hours at room temperature. Take a 5mL disposable syringe, remove the needle and absorb the Agrobacterium solution, and infiltrate the back of the leaves of common tobacco (5-6 weeks old or 4-6 true leaves). Infiltra...

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Abstract

The invention relates to the field of plant antiviral gene engineering, and discloses an attenuated vaccine for resisting potato X virus as well as a preparation method and application of the attenuated vaccine. The attenuated vaccine for resisting the potato X virus is based on a TVBMV attenuated mutant, an effective gene fragment capable of inducing cross protection on the potato X virus is embedded in the TVBMV attenuated mutant, and the effective gene fragment at least comprises an RdRp gene of the potato X virus, wherein the RdRp gene comprises an Rd1 gene, an Rd2 gene and an Rd3 gene, and one of the Rd1 gene, the Rd2 gene and the Rd3 gene is embedded into the TVBMV attenuated mutant. The attenuated vaccine for resisting the potato X virus is stable in effect, can play an effective cross protection role, remarkably reduces the damage of plants infected by a virulent strain of the potato X virus, delays the attack of the plants, and greatly reduces the loss.

Description

technical field [0001] The invention belongs to the field of plant antiviral genetic engineering, in particular to an attenuated vaccine against potato X virus, a preparation method and application thereof. Background technique [0002] Virus disease is an important disease on crops, causing huge losses to agricultural production. Due to the variety of crop virus diseases and the complicated transmission routes, there are no varieties with immunity or high resistance to virus diseases in production, and there are no drugs with specific effects on virus diseases on the market, so the prevention and control of virus diseases is very difficult. [0003] Tobacco virus disease has always been an important factor restricting the production of tobacco leaves in my country. The main tobacco varieties currently in production are not ideal for resistance to virus diseases, and the control of tobacco virus diseases mainly relies on agricultural control and chemical control. However, ...

Claims

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Application Information

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IPC IPC(8): C12N7/01C12N1/21A01N63/40A01P1/00C12R1/94
CPCC12N7/00C07K14/005A01N63/00C12N2770/00021C12N2770/00022C12N2770/00031
Inventor 李向东刘锦李现道耿超田延平刘茜郭兆奎万秀清乔婵李若
Owner SHANDONG AGRICULTURAL UNIVERSITY
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